The present invention relates to non-human animals, preferably mice, lacking the sno gene and cells thereof, preferably embryos thereof, or a method for screening substances by using the above animals or cells thereof.
A coactivator such as CBP, and a corepressor such as N-CoR, are called as xe2x80x9cmediating factorsxe2x80x9d. The mediating factors were identified as molecule which acted as a bridge between the transcriptional regulatory factor binding to the enhancer/silencer upstream of the promoter and a basal transcription factor, such as TBP, binding to the core promoter. The mediating factors form complexes with histone acetyltransferase (HAT, an enzyme acetylating histone) or, on the contrary, with histone deacetylase (HDAC, a deacetylating enzyme from histone), indicating that it changes the chromatin structure through acetylation of histone in order to regulate gene expression. The regulation of gene expression by modifying the chromatin structure is thought to be involved in various life processes such as immunity and development and differentiation, but the mechanisms are unknown.
Ikaros, which was originally thought to be a T cell specific transcriptional activation factor, has been demonstrated, by recent studies of a British group, to recruit a T cell specific gene into the heterochromatin region, which is the transcription inactivated region. It then plays a role in the expression of these genes in undifferentiated cells. As can be understood from this example, gene expression regulation mediated by the chromatin structure is very important in expression and differentiation of the immune system. Consequently, studies on the mediating factors provide a breakthrough in this area.
One of the important matters in discussing the physiological function of the mediating factors is xe2x80x9chaploinsufficiencyxe2x80x9d, which means that half is insufficient. Since eucaryotic cells are diploid and normally have two copies of genes, even if a mutation occurs in one copy of the gene, the phenotype does not appear in most cells. Studies by the inventors of the present invention have found that, in the case of the mediating factors, if a mutation occurs in one copy of the gene, the amount of gene product reduces to half. This process connects directly with an abnormal phenotype.
This leads to the idea that, since limited types and numbers of the mediating factors are commonly used for large numbers of the transcriptional regulatory factors, the reduction in the amount of gene product to half is directly related to the abnormal phenotype. Consequently, diseases which develop as a result of a mutation in the mediating factors gene commonly occur in the group of so-called xe2x80x9cautosomal dominant diseases (which mean onset occurs as a result of a mutation of one copy of the gene in an autosome)xe2x80x9d, and they frequently result in a disease.
N-CoR was originally identified as a corepressor which is essential for transcriptional regulation of the action of the intranuclear hormone receptor and, together with a factor such as Sin3 or HDAC, constitutes a large complex. We have analyzed the physiological function of ski/sno, a constitutional factor of the N-CoR corepressor complex.
The ski gene was originally found as an oncogene which transforms the chicken embryonic fibroblastoma cells (Stavnezer, E., et al., Mol. Cell Biol., 9, 4038-4945, 1989). We have found that the protein encoded by the c-ski gene and its related gene sno (Nomura, N., et al., Nucleic Acid Res., 17, 5489-5500, 1989) bind to the corepressor N-CoR/SMRT (Hoerlein, A. J., et al., Nature, 377, 397-404, 1995; Chen, J. D., et al., Nature, 377, 454-457, 1995), and to mSin3 (Ayer, D. E., et al., Cell, 80, 767-776, 1995; Screiber-Agus, N., et al., Cell, 80, 777-786, 1995), and form a complex with histone deacetylase (HDAC).
Ski/Sno is essential for the transcriptional repression which is mediated by thyroid hormone receptor and Mad. Further, these are directly bound with Rb as well as requiring an Rb-mediating repressive factor.
We have found that Ski, which was identified as an oncogene product, and its related gene product Sno, bind directly with N-CoR and Sin3 to function as the constitutional factor of the N-CoR complex. Quite interestingly, the transcriptional suppression generated by the tumor suppressive gene products Mad and Rb require Ski/Sno. Heterogeneous mice lacking Sno were prepared and a carcinogenesis experiment was conducted. As a result, we have found that these mice were highly cancer-prone, and it was shown for the first time that the Ski/Sno gene family was a tumor suppressor gene. In addition, heterogeneous mice lacking sno exhibit various abnormalities in their immune systems. The mice have extraordinarily high levels of induced Th1 group cytokines such as interferon xcex3, and easily develop ulcerative colitis. The germinal center does not form in these mice and induction of B cell proliferation by LPS is not observed. We have also examined the relationships between various abnormalities and diseases observed in mice in which the amount of Ski or Sno is reduced to half the normal level, and their molecular mechanisms.
An object of the present invention is to provide heterogeneous or homogeneous mouse variants lacking the sno gene and cells of these animals by constructing heterogeneous or homogeneous mouse variants lacking sno gene and analyzing functions of sno gene.
Another object of the present invention is to provide screening systems involving the use of these animals or their cells.
The present invention includes non-human animals, preferably mice, lacking sno genes and cells thereof. The animals lacking sno gene and cells thereof of the present invention include both heterogeneous variants and homogeneous variants.
Further, the present invention relates to a method for screening candidates for drugs by using the above animals or cells thereof.